Introduction:Visceral Leishmaniasis (VL), caused by Leishmania donovani, is a fatal disease, necessitating an effective vaccine. This study aims to develop a vaccine by evaluating the combination of recombinant kinesin protein (rKIN) with Bacille Calmette-Guerin (BCG) as an adjuvant against experimental VL. Materials and Methods:The immune response was analyzed against the purified rKIN of L. donovani with and without BCG adjuvant by using BALB/C mice. Mice were divided into 6 groups comprising of 6 animals in each group for the vaccine intramuscularly. All 6 Groups were immunized either with BCG, rKIN, or combination of the two, with different doses. Saline was used as negative control. Each group was further divided into 2 subgroups. One subgroup of animals from each group was challenged with L. donovani promastigotes 1 × 106 cells/100 μl per animal. The extent of protection was evaluated by estimating the reduction in the number of parasites in the spleen, quantity of nitric oxide (NO), reactive oxygen species (ROS) in the peritoneal cells, and production of cytokines in blood serum. Results:Significant parasite reduction (70%-90%) was observed in the spleens of groups receiving rKIN (50µg or 100µg) with BCG. NO and ROS production increased by 60%-95% and 70%-90%, respectively. The 100µg rKIN with BCG group demonstrated substantial protection (P < 0.001) with upregulated interferon-gamma (IFN-γ), tumor necrosis factor, interleukin-2 (IL-2), and downregulated IL-4, IL-10, and IL-17. Statistical analysis confirmed significant differences (P < 0.001) between vaccinated and control groups. Conclusions:The combination of 100µg rKIN with BCG shows potential as a vaccine candidate against VL.

The complex lipid composition of Mycobacterium tuberculosis (MTB) plays a pivotal role in pathogenesis, immune evasion, and antimicrobial resistance. The continued surge in drug-resistant strains underscores the need for a deeper understanding of the molecular architecture underlying MTB pathogenesis and drug resistance. In this study, a comparative lipidomics approach was applied to explore the resistance-associated lipid alterations in drug-sensitive (DS), drug-resistant (DR), multidrug-resistant (MDR), and pre-extensively drug-resistant (PXDR) MTB clinical isolates. Lipids derived from whole bacilli (TL) and cell wall (CWL) extracts were separately analyzed using untargeted and targeted lipidomics approaches. Untargeted analysis revealed the abundance of fatty acyls, glycerolipids, glycerophospholipids, prenol lipids, polyketides, and saccharolipids, with distinct phenotypes and compartment-specific distributions. Notably, CWL extracts showed clearer resistance-associated separations relative to TL extracts. Targeted profiling further demonstrated enrichment of glycerolipids and PC and LPC lipids among drug-resistant isolates. Biomarker analysis identified discriminative lipid species in both extracts, albeit with greater discriminatory power in CWL. Overall, these findings elucidate coordinated and compartment-associated lipid alterations at the species level in different MTB clinical isolates. This will help to provide a valuable source for screening diagnostic biosignatures and intersecting biosynthetic pathways of mycobacterial lipids in the evolution of drug resistance in MTB for therapeutic interventions.

Recently, the World Health Organization (WHO) endorsed three new diagnostic strategies for tuberculosis (TB): Near-point-of-care nucleic acid amplification tests (NPOC-NAATs), tongue swabs as an alternative to sputum, and pooled sputum testing. Each addresses real barriers to case detection in high-burden settings. However, the supporting evidence for each remains thin precisely in the populations where the diagnostic gap is widest: children, people living with HIV (PLHIV), and patients with extrapulmonary or paucibacillary disease. We argue that these recommendations should be viewed as the start of an evidence-generation cycle rather than its endpoint and propose four concrete safeguards before unrestricted programmatic scale-up.

Accurate and rapid diagnosis is crucial for starting effective treatment for tuberculosis (TB) and mitigating the transmission. Globally, nearly one-third of all TB cases remain undetected each year and consequently these are not reported. On top of that, the emergence of drug-resistant TB poses an added challenge. In the past 15 years, several advances have been made for improved diagnosis, including liquid culture and drug susceptibility, line probe assay for drug resistance detection, and cartridge-based nucleic acid amplification tests for rapid diagnosis of TB and drug resistance detection. However, some challenges remain, despite the clear edge of these new advances over the age-old conventional methods. Despite these advances, accurate, affordable, and accessible diagnosis of TB remains a challenge, especially in rural and difficult-to-reach settings, where the most desirable test would be a point-of-care triage test. Nevertheless, several attempts are being made in this direction, and in this article, we review these research advances that can help the TB elimination from India.

Diagnosis of Tuberculosis (TB) is time-consuming, cumbersome, and expensive. Moreover, there is a lack of real-time monitoring of screening and testing as well as data management and storage. Serological screening point-of-care tests, which are rapid and affordable, have been viewed as a desirable method for TB diagnosis for a long time, although they cannot be used to confirm the disease. Three novel antigens of Mycobacterium tuberculosis (MTB), the causative agent of TB, have been considered for the colorimetric diagnosis. The immunochromatic flow-through test (ICT) devices were developed to screen the suspected cases of active TB with high sensitivity and specificity. In this work, using these ICT devices, we have now developed an image sensing method based on dataset of images and trained a model to create a custom-made phone application for the accurate detection of TB with real-time reporting. The image sensing of the colorimetric outcome was integrated with different classifications, of which Feedforward Neural Network (FNN) allowed us to make predictions with an overall accuracy of ~82%, and this is on par with the results of existing literature. With a sensitivity of 87%, specificity of 82%, AUC score of 0.84, and an F1-score of 81% (No TB) and 82% (with TB), the suggested approach demonstrates enhanced efficiency compared to naked eye results. This image sensing technique can significantly reduce the possibility of errors resulting from visual results, and color ambiguity.

Rapid detection of infectious diseases like COVID-19, flu, and dengue is crucial for healthcare professionals preparing for contagious outbreaks. Given the constant mutations in viruses and the recurring emergence of threats like Nipah and Zika, there is an urgent demand for a technology capable of distinguishing between infections that share similar symptoms. In this paper, we utilize laser-based Raman scattered signals from a drop of dried blood plasma, combined with generative artificial intelligence, to provide a rapid and precise diagnosis. Our optimized model exhibits exceptional performance, yielding high predictive scores of 96%, 98%, and 100% for flu, COVID-19, and dengue, respectively. The proposed Raman spectroscopic analysis, with a rapid turnaround time, can ensure a near-accurate diagnosis and proper quarantining of highly infectious cases. Furthermore, the potential extension of our method to include other viral diseases offers an alternative to the challenge of developing different diagnostic kits for each disease.

Tuberculosis (TB) is still a major health concern. However, each year more than one-third of all global TB cases remain undetected and unreported. On top of that, emergence of drug-resistant TB poses a major challenge. Therefore, a Reliable, Accessible, Cost-Effective, and Easy (RACE) diagnostic modality is crucial for starting suitable treatment of TB and curtailing its transmission. In the last two decades, several advances have been made for improved diagnosis, which include liquid culture and drug susceptibility testing (DST), line probe assay (LPA) for drug resistance detection at the molecular level, and cartridge-based nucleic acid amplification tests (CBNAAT) for rapid diagnosis of TB and rifampicin resistance detection. Newer drugs and treatment regimens have been introduced and vaccines are in the pipeline. Despite these advances and opportunities, a precise, affordable, and accessible diagnostic model is yet to be evolved, especially in rural and difficult-to-reach areas, where the most desirable test would be a test that is easy to perform, accessible to masses, is cost-effective, besides being reliable. Only a point-of-care triage test can meet these requirements, which can be used by an unskilled or minimally trained healthcare worker or even by the patient (self-testing). This test should be able to detect all forms of tuberculosis and latent TB infection. Currently, no such test is available. In this narrative review, we will discuss how such a diagnostic modality can help eliminate TB.

BACKGROUND:Extrapulmonary tuberculosis (EPTB) poses a diagnostic challenge due to its paucibacillary nature and low diagnostic yield of traditional methods. Molecular diagnostic tools like Xpert MTB/RIF, Xpert MTB/RIF Ultra, and Truenat MTB offer promising alternatives for the rapid and higher detection rates for pulmonary TB samples. However, the yield of these newly introduced molecular tests on extra-pulmonary samples requires country-specific evaluation and comparative analysis. METHODOLOGY:This study evaluated the diagnostic performance of Xpert MTB/RIF, Xpert MTB/RIF Ultra, and Truenat MTB assays in detecting EPTB on 211 clinical specimens collected at a tertiary care centre of central India. The assays' sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPP) were compared against a composite reference standard. RESULTS:Xpert MTB/RIF Ultra exhibited the highest sensitivity (50%) for EPTB detection, outperforming the standard GeneXpert MTB/RIF (29.4%) and Truenat MTB (35.3%), with comparable specificity across all three assays. Among the specimen types, pus samples demonstrated the highest (value) diagnostic yield. These findings highlight the superior diagnostic capabilities of Xpert MTB/RIF Ultra, particularly in paucibacillary cases. CONCLUSION:Xpert Ultra demonstrates superior sensitivity for EPTB detection compared to Xpert MTB/RIF and Truenat MTB, making it a promising tool to enhance diagnostic accuracy in paucibacillary TB. Its adoption as a standard diagnostic method could significantly improve EPTB management. These findings advocate for integrating advanced molecular diagnostics into routine tuberculosis workflows to reduce diagnostic delays and improve patient outcomes. CLINICAL TRAIL NUMBER:Not applicable.

Toxoplasma gondii is a widespread protozoan parasite that poses significant health risks globally. Current serological tests for diagnosing T. gondii infections are hindered by high costs, cumbersomeness, and the necessity for skills and expertise. This study aimed to identify promising antigens for the development of an immunochromatographic rapid diagnostic test (RDT). Whole-cell antigens were isolated from the virulent RH strain of T. gondii maintained in Swiss albino male mice. After infection, peritoneal fluid was harvested, and tachyzoites were processed to obtain whole cell lysates, which were subjected to SDS-PAGE and Western blot analysis. Five novel antigenic protein bands reactive to anti-Toxoplasma IgG and IgM antibodies were identified on western blot. Subsequent LC–MS/MS analysis revealed 158 proteins. However, only 18 proteins were selected on the basis of high mascot score (> 40) and were investigated further. On BLAST search 10 of these proteins exhibited significant homology (> 90

Objectives:Rising antimicrobial resistance (AMR) in Escherichia coli urinary tract infections (UTI) poses a global challenge. Evidence-based treatment of cystitis requires local resistance data. The DASH to Protect Antibiotics (https://dashuti.com/), a multi-regional group, supports centers in generating and sharing focused antibiograms to guide stewardship in community UTIs. This multi-country study aimed to describe antimicrobial susceptibility patterns of community-acquired E. coli isolates in low, middle, and high-income countries (LMICs and HICs). Methods:The study was conducted in 37 representative centers across 13 countries in Asia (Middle East and Indian Subcontinent), Africa, Europe, and North America. A rigorous comparative analysis of the antimicrobial susceptibility of E. coli isolated from cases of simple cystitis presenting in outpatient or emergency departments was carried out. The impact of gross domestic product, climate, and population density per km2 on E. coli susceptibility profile was analyzed using the Kruskal-Wallis test and two-way analysis of variance. Results:Antimicrobial susceptibility varied significantly between LMICs and HICs, with nitrofurantoin (89%) and fosfomycin (96%) emerging as empiric choices globally. Across most centers, susceptibility to other oral antimicrobials was low: co-trimoxazole <60%, amoxicillin-clavulanic acid <70%, first-generation cephalosporins <50%, fluoroquinolones <60%. Injectable antibiotics fared better: piperacillin-tazobactam >70%, amikacin and meropenem >80%. Higher susceptibilities were noted in countries with high gross domestic product (P < 0.001) and humidity (P = 0.002). Conclusion:Marked geographical differences in E. coli susceptibility patterns support the need for localized antibiograms and tailored empirical therapy. This study reinforces the utility of nitrofurantoin and fosfomycin as first-line agents and discourages the use of fluoroquinolones and third-generation cephalosporins.

ABSTRACT The objective of this study is to correlate rpoB mutations found on the new-generation sequencing (NGS) in Mycobacterium tuberculosis (MTB) isolates with minimum inhibitory concentrations (MICs) to the rifampicin (RIF). We assessed the minimum inhibitory concentrations for 151 archived clinical MTB isolates that were determined phenotypically susceptible to RIF (101; 66.89%), and the remaining 50 (50; 33.11%) were resistant to RIF by BACTEC MGIT SIRE DST. MIC values were determined using the colorimetric redox indicator (Resazurin/REMA) method, and results were correlated with rpoB gene mutations associated with rifampicin resistance found. Comparing the MIC and critical concentration, we found that 15 of these 101 (14.85%) isolates were misclassified by MGIT-960 as sensitive at standard critical concentration (1.0 µg/mL) though these were found to have low-level RIF resistance by CRI assay (MIC 0.50 to 1.0µg/mL) and NGS. We found that all 15 isolates contained non-synonymous mutations, the commonest being the Ile572Phe (7, 46.66%), followed by Leu533Pro (3, 20.0%), His526Leu (2, 13.33%), His526Asn+Ile572 Phe (1), Asp516Tyr (1), and Leu533Pro+Pro564 Arg (1). These mutations are reported to confer low-level RIF resistance. But we did not find any mutation at MIC ≤0.25 µg/mL. We found that a significant number of MTB isolates have phenotypic and genotypic discordance. Taking 1.0 µg/mL of rifampicin as a critical concentration, isolates from approximately 15% of patients are misidentified as susceptible to rifampicin, even when these strains carry low-level drug resistance-conferring mutations and have the potential to develop clinical multidrug-resistant tuberculosis.IMPORTANCETuberculosis (TB) remains a leading cause of morbidity and mortality worldwide, killing millions every year. The emergence of multidrug-resistant and extensively drug-resistant TB forms poses a challenge to the TB control programs. In the past few decades, several molecular tests for rapid detection and drug resistance determination have been developed. But these can miss the genetic mutations that confer low-level resistance to rifampicin (RIF), a critical constituent for treating drug-susceptible TB. On the other hand, for the phenotypic methods, a cutoff value is fixed, known as critical concentration (CC). The current WHO-endorsed CC for rifampicin is 1.0 μg/mL in liquid culture for confirmation of drug resistance; because of that in this system too, low-level RIF resistance may not be correctly identified. Therefore, it is important that either the CC for phenotypic methods is lowered or the specific mutations are included in the molecular tests. This study provides important insights in that direction.

Zoonotic tuberculosis is a neglected subject that has the potential to impede the effectiveness of the TB elimination program. The present study aimed to determine the genetic diversity and drug resistance in the Mycobacterium tuberculosis isolates from captive wild animals. A total of 67 tissue samples were collected from 33 animals, comprising 21 wild captive animals from various species and 12 slaughtered domestic buffaloes. These samples were subjected to the detection of Mycobacterial species by culture isolation, and further molecular identification by mPCR, Xpert-Ultra and TrueNat MTB/Rif assay; followed by drug susceptibility profiling by MTBDRplus and Spoligotyping of the isolates. Of the 67 samples from captive zoo animals, 44 samples were culture positive. Of these, 38 isolates were identified as Mycobacterium tuberculosis Complex (MTBC) and remaining 6 were identified as non- tuberculous mycobacteria (NTM). All NTM isolates were from different tissues of a Tigress which also had mixed infection with MTBC. All the 38 culture isolates were further subjected to phenotypic drug susceptibility testing (pDST) and genotyping. Twenty-eight (73.69%) of them, were pan-susceptible, 9 (23.68%) exhibited isoniazid mono-resistance, and 1 (2.63%) was rifampicin mono-resistant. On genotyping, 27 (71.05%) of the samples were classified as 'Orphan'. Ten (26.32%) isolates were identified as CAS1_DELHI, clustered within SIT number 375, while one sample (2.63%) remained unidentified. The drug resistance and genotyping patterns were similar to the human population. Our results show that M. tuberculosis was major cause of Zoonotic TB and should be considered as potential reverse zoonotic agent in India.

AIM:This study was undertaken to compare the proteomic profile of sequential isolates of Beijing lineage Mycobacterium tuberculosis (M. tuberculosis) from a patient who developed drugresistant tuberculosis (TB) in vivo during anti-tuberculosis therapy (ATT). INTRODUCTION:Various studies have found the Beijing lineage of M. tuberculosis strongly associated with multidrug resistance (MDR) development. OBJECTIVES:To identify and characterize the differentially expressed proteins during the in-vivo drug resistance conversion in M. tuberculosis Beijing lineage clinical isolates. METHODS:Drug-susceptible and drug-resistant M. tuberculosis isolates were confirmed as Beijing lineage. The isolates were grown in Middlebrook 7H9 medium for two weeks, and whole-cell lysate was prepared. Two-dimensional gel electrophoresis (2DGE) was used for proteomic analysis, and differentially expressed proteins were identified using MALDI-TOF-MS. Bioinformatics tools were used for molecular docking, phosphorylation, and pupylation site prediction. RESULTS:Seventeen proteins were found overexpressed in drug-resistant isolates as compared to drugsusceptible isolates, including the six proteins with unknown functions. Molecular docking showed that Isoniazid (INH) and Rifampicin (RIF) interacted with their conserved domains/active sites of these proteins. DISCUSSION:We characterized two paired clinical isolates from a patient, one being INH and RIF susceptible and other resistant. The comparative analysis of over expressed proteins showed that 5 of 17 proteins belonged to the cell wall and cell processes functional group, 3 to virulence, detoxification, adaptation functional group, and 3 to information pathways functional group, 2 proteins belonged to insertion sequences and phage functional group, and 1 each (Rv0242c, Rv2970c and Rv3208A) to lipid metabolism, intermediary metabolism & respiration and regulatory functional group. We found that the Rv1827, Rv2626c, Rv2714, Rv2970c, Rv3208A, and Rv3881c proteins showed significant interaction in-silico with INH and RIF. CONCLUSIONS:These over-expressed proteins probably play an important role in drug resistance development, and further studies on drug resistance mechanisms could provide more details. We also believe that these over-expressed proteins could be used as biomarkers for early prediction of in-vivo drug-resistance development.